Polypeptides having cellobiohydrolase II activity and polynucleotides encoding same

ABSTRACT

The present invention relates to polypeptides having cellobiohydrolase II activity and polynucleotides having a nucleotide sequence which encodes for the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid constructs as well as methods for producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 10/540,091filed Jun. 20, 2005, (now U.S. Pat. No. 7,348,168), which is a 35 U.S.C.371 national application of PCT/DK2003/000914 filed Dec. 19, 2003, whichclaims priority or the benefit under 35 U.S.C. 119 of U.S. provisionalapplication No. 60/435,100 filed Dec. 20, 2002, the contents of whichare fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to polypeptides having cellobiohydrolaseII (also referred to as CBH II or CBH 2) activity and polynucleotideshaving a nucleotide sequence which encodes for the polypeptides. Theinvention also relates to nucleic acid constructs, vectors, and hostcells comprising the nucleic acid constructs as well as methods forproducing and using the polypeptides.

BACKGROUND OF THE INVENTION

Cellulose is an important industrial raw material and a source ofrenewable energy. The physical structure and morphology of nativecellulose are complex and the fine details of its structure have beendifficult to determine experimentally. However, the chemical compositionof cellulose is simple, consisting of D-glucose residues linked bybeta-1,4-glycosidic bonds to form linear polymers with chains length ofover 10,000 glycosidic residues.

In order to be efficient, the digestion of cellulose requires severaltypes of enzymes acting cooperatively. At least three categories ofenzymes are necessary to convert cellulose into glucose: endo(1,4)-beta-D-glucanases (EC 3.2.1.4) that cut the cellulose chains atrandom; cellobiohydrolases (EC 3.2.1.91) which cleave cellobiosyl unitsfrom the cellulose chain ends and beta-glucosidases (EC 3.2.1.21) thatconvert cellobiose and soluble cellodextrins into glucose. Among thesethree categories of enzymes involved in the biodegradation of cellulose,cellobiohydrolases are the key enzymes for the degradation of nativecrystalline cellulose.

Exo-cellobiohydrolases (Cellobiohydrolase II or CBH II) refer to thecellobiohydrolases which degrade cellulose by hydrolyzing the cellobiosefrom the non-reducing end of the cellulose polymer chains. Thecellobiohydrolase II group belongs to the same EC group, i.e., EC3.2.1.91, as the cellobiohydrolase I group, the difference being thatcellobiohydrolase I degrade cellulose by hydrolyzing the cellobiose fromthe reducing end of the cellulose polymer chains.

It is an object of the present invention to provide improvedpolypeptides having cellobiohydrolase II activity and polynucleotidesencoding the polypeptides. The improved polypeptides may have improvedspecific activity and/or improved stability—in particular improvedthermostability. The polypeptides may also have an improved ability toresist inhibition by cellobiose.

SUMMARY OF THE INVENTION

In a first aspect the present invention relates to a polypeptide havingcellobiohydrolase 11 activity, selected from the group consisting of:

(a) a polypeptide comprising an amino acid sequence selected from thegroup consisting of:

-   -   an amino acid sequence which has at least 75%, identity with the        amino acid sequence shown as amino acids 1 to 477 of SEQ ID NO:        2,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 85% identity with the partial amino acid sequence shown as        amino acids 1 to 82 of SEQ ID NO: 4,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 85% identity with the partial amino acid sequence shown as        amino acids 1 to 420 of SEQ ID NO: 4,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 80% identity with the partial amino acid sequence shown as        amino acids 1 to 139 of SEQ ID NO: 6,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 95% identity with the partial amino acid sequence shown as        amino acids 1 to 102 of SEQ ID NO: 8,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 85% identity with the partial amino acid sequence shown as        amino acids 1 to 144 of SEQ ID NO: 10,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 75% identity with the partial amino acid sequence shown as        amino acids 1 to 99 of SEQ ID NO: 12,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 85% identity with the partial amino acid sequence shown as        amino acids 1 to 140 of SEQ ID NO: 14,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 75% identity with the partial amino acid sequence shown as        amino acids 1 to 109 of SEQ ID NO: 16,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 75% identity with the amino acid sequence shown as SEQ ID        NO: 16,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 75% identity with the partial amino acid sequence shown as        amino acids 1 to 143 of SEQ ID NO: 18,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 70% identity with the partial amino acid sequence shown as        amino acids 1 to 71 of SEQ ID NO: 20,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 60% identity with the amino acid sequence shown as amino        acids 1 to 220 of SEQ ID NO: 22,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 65% identity with the amino acid sequence shown as amino        acids 1 to 458 of SEQ ID NO: 24, and    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 70% identity with the amino acid sequence shown as amino        acids 1 to 390 of SEQ ID NO: 26;

(b) a polypeptide comprising an amino acid sequence selected from thegroup consisting of:

-   -   an amino acid sequence which has at least 75% identity with the        polypeptide encoded by the cellobiohydrolase II encoding part of        the nucleotide sequence present in Chaetomium thermophilum,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 85% identity with the polypeptide encoded by the        cellobiohydrolase II encoding part of the nucleotide sequence        present in Myceliophtora thermophila,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 80% identity with the polypeptide encoded by    -   an amino acid sequence which has at least 80% identity with the        polypeptide encoded by the cellobiohydrolase II encoding part of        the nucleotide sequence present in Acremonium thermophilum,    -   an amino acid sequence which has at least 95% identity with the        polypeptide encoded by the cellobiohydrolase II encoding part of        the nucleotide sequence present in Melanocarpus sp.,    -   an amino acid sequence which has at least 85% identity with the        polypeptide encoded by the cellobiohydrolase II encoding part of        the nucleotide sequence present in Thielavia microspora,    -   an amino acid sequence which has at least 75% identity with the        polypeptide encoded by the cellobiohydrolase II encoding part of        the nucleotide sequence present in Aspergillus sp.,    -   an amino acid sequence which has at least 85% identity with the        polypeptide encoded by the cellobiohydrolase II encoding part of        the nucleotide sequence present in Thielavia australiensis,    -   an amino acid sequence which has at least 75% identity with the        polypeptide encoded by the cellobiohydrolase II encoding part of        the nucleotide sequence present in Aspergillus tubingensis,    -   an amino acid sequence which has at least 75% identity with the        polypeptide encoded by the cellobiohydrolase II encoding part of        the nucleotide sequence present in Gloeophyllum trabeum,    -   an amino acid sequence which has at least 70% identity with the        polypeptide encoded by the cellobiohydrolase II encoding part of        the nucleotide sequence present in Meripilus giganteus,    -   an amino acid sequence which has at least 60% identity with the        polypeptide encoded by the cellobiohydrolase II encoding part of        the nucleotide sequence present in Trichophaea saccata,    -   an amino acid sequence which has at least 65% identity with the        polypeptide encoded by the cellobiohydrolase II encoding part of        the nucleotide sequence present in Stilbella annulata, and    -   an amino acid sequence which has at least 70% identity with the        polypeptide encoded by the cellobiohydrolase II encoding part of        the nucleotide sequence present in Malbrancheae cinnamomeal;

(c) a polypeptide comprising an amino acid sequence selected from thegroup consisting of:

-   -   an amino acid sequence which has at least 75% identity with the        polypeptide encoded by nucleotides 63 to 1493 of SEQ ID NO: 1,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 85% identity with the polypeptide encoded by nucleotides 1        to 246 of SEQ ID NO: 3,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 85% identity with the polypeptide encoded by nucleotides 1        to 1272 of SEQ ID NO: 3,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 80% identity with the polypeptide encoded by nucleotides 1        to 417 of SEQ ID NO: 5,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 95% identity with the polypeptide encoded by nucleotides 1        to 306 of SEQ ID NO: 7,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 85% identity with the polypeptide encoded by nucleotides 1        to 432 of SEQ ID NO: 9,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 75% identity with the polypeptide encoded by nucleotides 1        to 297 of SEQ ID NO: 11,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 85% identity with the polypeptide encoded by nucleotides 1        to 420 of SEQ ID NO: 13,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 75% identity with the polypeptide encoded by nucleotides 1        to 330 of SEQ ID NO: 15,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 75% identity with the polypeptide encoded by nucleotides 1        to 1221 of SEQ ID NO: 15,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 75% identity with the polypeptide encoded by nucleotides 1        to 1221 of SEQ ID NO: 15,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 75% identity with the polypeptide encoded by nucleotides 1        to 429 of SEQ ID NO: 17,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 70% identity with the polypeptide encoded by nucleotides 1        to 213 of SEQ ID NO: 19,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 60% identity with the polypeptide encoded by nucleotides        43 to 701 of SEQ ID NO: 21,    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 65% identity with the polypeptide encoded by nucleotides        21 to 1394 of SEQ ID NO: 23, and    -   a polypeptide comprising an amino acid sequence selected from        the group consisting of: an amino acid sequence which has at        least 70% identity with the polypeptide encoded by nucleotides        41 to 1210 of SEQ ID NO: 25;

(d) a polypeptide which is encoded by a nucleotide sequence whichhybridizes under high stringency conditions with a polynucleotide probeselected from the group consisting of

-   -   (i) the complementary strand of the nucleotides selected from        the group consisting of        -   nucleotides 63 to 1493 of SEQ ID NO: 1,        -   nucleotides 1 to 246 of SEQ ID NO: 3,        -   nucleotides 1 to 1272 of SEQ ID NO: 3,        -   nucleotides 1 to 417 of SEQ ID NO: 5,        -   nucleotides 1 to 306 of SEQ ID NO: 7,        -   nucleotides 1 to 432 of SEQ ID NO: 9,        -   nucleotides 1 to 297 of SEQ ID NO: 11,        -   nucleotides 1 to 420 of SEQ ID NO: 13,        -   nucleotides 1 to 330 of SEQ ID NO: 15,        -   nucleotides 1 to 1221 of SEQ ID NO: 15,        -   nucleotides 1 to 429 of SEQ ID NO: 17,        -   nucleotides 1 to 213 of SEQ ID NO: 19,        -   nucleotides 43 to 701 of SEQ ID NO: 21,        -   nucleotides 21 to 1394 of SEQ ID NO: 23, and        -   nucleotides 41 to 1210 of SEQ ID NO: 25;    -   (ii) the complementary strand of the nucleotides selected from        the group consisting of        -   nucleotides 63 to 563 of SEQ ID NO: 1,        -   nucleotides 43 to 543 of SEQ ID NO: 21,        -   nucleotides 21 to 521 of SEQ ID NO: 23, and        -   nucleotides 41 to 541 of SEQ ID NO: 25;    -   (iii) the complementary strand of the nucleotides selected from        the group consisting of:        -   nucleotides 63 to 263 of SEQ ID NO: 1,        -   nucleotides 1 to 200 of SEQ ID NO: 3,        -   nucleotides 1 to 200 of SEQ ID NO: 5,        -   nucleotides 1 to 200 of SEQ ID NO: 7,        -   nucleotides 1 to 200 of SEQ ID NO: 9,        -   nucleotides 1 to 200 of SEQ ID NO: 11,        -   nucleotides 1 to 200 of SEQ ID NO: 13,        -   nucleotides 1 to 200 of SEQ ID NO: 15,        -   nucleotides 1 to 1221 of SEQ ID NO: 15,        -   nucleotides 1 to 200 of SEQ ID NO: 17,        -   nucleotides 1 to 200 of SEQ ID NO: 19,        -   nucleotides 43 to 243 of SEQ ID NO: 21,        -   nucleotides 21 to 221 of SEQ ID NO: 23, and        -   nucleotides 41 to 241 of SEQ ID NO: 25; and

(e) a fragment of (a), (b) or (c) that has cellobiohydrolase IIactivity.

In a second aspect the present invention relates to a polynucleotidehaving a nucleotide sequence which encodes for the polypeptide of theinvention.

In a third aspect the present invention relates to a nucleic acidconstruct comprising the nucleotide sequence, which encodes for thepolypeptide of the invention, operably linked to one or more controlsequences that direct the production of the polypeptide in a suitablehost.

In a fourth aspect the present invention relates to a recombinantexpression vector comprising the nucleic acid construct of theinvention.

In a fifth aspect the present invention relates to a recombinant hostcell comprising the nucleic acid construct of the invention.

In a sixth aspect the present invention relates to a method forproducing a polypeptide of the invention, the method comprising:

(a) cultivating a strain, which in its wild-type form is capable ofproducing the polypeptide, to produce the polypeptide; and

(b) recovering the polypeptide.

In a seventh aspect the present invention relates to a method forproducing a polypeptide of the invention, the method comprising:

(a) cultivating a recombinant host cell of the invention underconditions conducive for production of the polypeptide; and

(b) recovering the polypeptide.

In an eight aspect the present invention relates to a method for in-situproduction of a polypeptide of the invention, the method comprising:

(a) cultivating a recombinant host cell of the invention underconditions conducive for production of the polypeptide; and

(b) contacting the polypeptide with a desired substrate without priorrecovery of the polypeptide.

Other aspects of the present invention will be apparent from the belowdescription and from the appended claims.

DEFINITIONS

Prior to discussing the present invention in further details, thefollowing terms and conventions will first be defined:

Substantially pure polypeptide: In the present context, the term“substantially pure polypeptide” means a polypeptide preparation whichcontains at the most 10% by weight of other polypeptide material withwhich it is natively associated (lower percentages of other polypeptidematerial are preferred, e.g., at the most 8% by weight, at the most 6%by weight, at the most 5% by weight, at the most 4% at the most 3% byweight, at the most 2% by weight, at the most 1% by weight, and at themost 0.5% by weight). Thus, it is preferred that the substantially purepolypeptide is at least 92% pure, i.e., that the polypeptide constitutesat least 92% by weight of the total polypeptide material present in thepreparation, and higher percentages are preferred such as at least 94%pure, at least 95% pure, at least 96% pure, at least 96% pure, at least97% pure, at least 98% pure, at least 99%, and at the most 99.5% pure.The polypeptides disclosed herein are preferably in a substantially pureform. In particular, it is preferred that the polypeptides disclosedherein are in “essentially pure form”, i.e., that the polypeptidepreparation is essentially free of other polypeptide material with whichit is natively associated. This can be accomplished, for example, bypreparing the polypeptide by means of well-known recombinant methods.Herein, the term “substantially pure polypeptide” is synonymous with theterms “isolated polypeptide” and “polypeptide in isolated form”.

Cellobiohydrolase II activity: The term “cellobiohydrolase II activity”is defined herein as a cellulose 1,4-beta-cellobiosidase (also referredto as Exo-glucanase, Exo-cellobiohydrolase or1,4-beta-cellobiohydrolase) activity, as defined in the enzyme class EC3.2.1.91 or CAZy Family Glycoside Hydrolase Family 6, which catalyzesthe hydrolysis of 1,4-beta-D-glucosidic linkages in cellulose andcellotetraose, releasing cellobiose from the non-reducing ends of thechains.

For purposes of the present invention, cellobiohydrolase II activity maybe determined according to the procedure described in Example 6.

In an embodiment, cellobiohydrolase II activity may be determinedaccording to the procedure described in Deshpande M V et al., Methods inEnzymology, pp. 126-130 (1988): “Selective Assay forExo-1,4-Beta-Glucanases”. According to this procedure, one unit ofcellobiohydrolase II activity (agluconic bond cleavage activity) isdefined as 1.0 micromole of p-nitrophenol produced per minute at 50° C.,pH 5.0. The polypeptides of the present invention should preferably haveat least 20% of the cellobiohydrolase II activity of a polypeptideconsisting of an amino acid sequence selected from the group consistingof SEQ ID NO: 2, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26. In aparticular preferred embodiment the polypeptides should have at least40%, such as at least 50%, preferably at least 60%, such as at least70%, more preferably at least 80%, such as at least 90%, most preferablyat least 95%, such as about or at least 100% of the cellobiohydrolase IIactivity of the polypeptide consisting of the amino acid sequenceselected from the group consisting of:

amino acids 1 to 477 of SEQ ID NO: 2,

amino acids 1 to 82 of SEQ ID NO: 4,

amino acids 1 to 420 of SEQ ID NO: 4,

amino acids 1 to 139 of SEQ ID NO: 6,

amino acids 1 to 102 of SEQ ID NO: 8,

amino acids 1 to 144 of SEQ ID NO: 10,

amino acids 1 to 99 of SEQ ID NO: 12,

amino acids 1 to 140 of SEQ ID NO: 14,

amino acids 1 to 109 of SEQ ID NO: 16,

amino acids 1 to 407 of SEQ ID NO: 16,

amino acids 1 to 143 of SEQ ID NO: 18,

amino acids 1 to 71 of SEQ ID NO: 20,

amino acids 1 to 220 of SEQ ID NO: 22,

amino acids 1 to 458 of SEQ ID NO: 24, and

amino acids 1 to 390 of SEQ ID NO: 26.

Identity: In the present context, the homology between two amino acidsequences or between two nucleotide sequences is described by theparameter “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined by using the program FASTAincluded in version 2.0× of the FASTA program package (see Pearson andLipman, 1988, “Improved Tools for Biological Sequence Analysis”, PNAS85:2444-2448; and Pearson, 1990, “Rapid and Sensitive SequenceComparison with FASTP and FASTA”, Methods in Enzymology 183:63-98). Thescoring matrix used was BLOSUM50, gap penalty was −12, and gap extensionpenalty was −2.

The degree of identity between two nucleotide sequences is determinedusing the same algorithm and software package as described above. Thescoring matrix used was the identity matrix, gap penalty was −16, andgap extension penalty was −4.

Fragment: When used herein, a “fragment” of a sequence selected from thegroup consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ IDNO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26is a polypeptide having one or more amino acids deleted from the aminoand/or carboxyl terminus of this amino acid sequence.

Allelic variant: In the present context, the term “allelic variant”denotes any of two or more alternative forms of a gene occupying thesame chromosomal locus. Allelic variation arises naturally throughmutation, and may result in polymorphism within populations. Genemutations can be silent (no change in the encoded polypeptide) or mayencode polypeptides having altered amino acid sequences. An allelicvariant of a polypeptide is a polypeptide encoded by an allelic variantof a gene.

Substantially sure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparation,wherein the polynucleotide has been removed from its natural geneticmilieu, and is thus free of other extraneous or unwanted codingsequences and is in a form suitable for use within geneticallyengineered protein production systems. Thus, a substantially purepolynucleotide contains at the most 10% by weight of otherpolynucleotide material with which it is natively associated (lowerpercentages of other polynucleotide material are preferred, e.g., at themost 8% by weight, at the most 6% by weight, at the most 5% by weight,at the most 4% at the most 3% by weight, at the most 2% by weight, atthe most 1% by weight, and at the most 0.5% by weight). A substantiallypure polynucleotide may, however, include naturally occurring 5′ and 3′untranslated regions, such as promoters and terminators. It is preferredthat the substantially pure polynucleotide is at least 92% pure, i.e.,that the polynucleotide constitutes at least 92% by weight of the totalpolynucleotide material present in the preparation, and higherpercentages are preferred such as at least 94% pure, at least 95% pure,at least 96% pure, at least 96% pure, at least 97% pure, at least 98%pure, at least 99%, and at the most 99.5% pure. The polynucleotidesdisclosed herein are preferably in a substantially pure form. Inparticular, it is preferred that the polynucleotides disclosed hereinare in “essentially pure form”, i.e., that the polynucleotidepreparation is essentially free of other polynucleotide material withwhich it is natively associated. Herein, the term “substantially purepolynucleotide” is synonymous with the terms “isolated polynucleotide”and “polynucleotide in isolated form”.

Modification(s): In the context of the present invention the term“modification(s)” is intended to mean any chemical modification of apolypeptide consisting of an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26, aswell as genetic manipulation of the DNA encoding that polypeptide. Themodification(s) can be replacement(s) of the amino acid side chain(s),substitution(s), deletion(s) and/or insertions(s) in or at the aminoacid(s) of interest.

Artificial variant: When used herein, the term “artificial variant”means a polypeptide having cellobiohydrolase II activity, which has beenproduced by an organism which is expressing a modified gene as comparedto SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25. The modified gene,from which said variant is produced when expressed in a suitable host,is obtained through human intervention by modification of a nucleotidesequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ IDNO: 23, and SEQ ID NO: 25.

cDNA: The term “cDNA” when used in the present context, is intended tocover a DNA molecule which can be prepared by reverse transcription froma mature, spliced, mRNA molecule derived from a eukaryotic cell. cDNAlacks the intron sequences that are usually present in the correspondinggenomic DNA. The initial, primary RNA transcript is a precursor to mRNAand it goes through a series of processing events before appearing asmature spliced mRNA. These events include the removal of intronsequences by a process called splicing. When cDNA is derived from mRNAit therefore lacks intron sequences.

Nucleic acid construct: When used herein, the term “nucleic acidconstruct” means a nucleic acid molecule, either single- ordouble-stranded, which is isolated from a naturally occurring gene orwhich has been modified to contain segments of nucleic acids in a mannerthat would not otherwise exist in nature. The term nucleic acidconstruct is synonymous with the term “expression cassette” when thenucleic acid construct contains the control sequences required forexpression of a coding sequence of the present invention.

Control sequence: The term “control sequences” is defined herein toinclude all components, which are necessary or advantageous for theexpression of a polypeptide of the present invention. Each controlsequence may be native or foreign to the nucleotide sequence encodingthe polypeptide. Such control sequences include, but are not limited to,a leader, polyadenylation sequence, propeptide sequence, promoter,signal peptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleotide sequence encoding a polypeptide.

Operably linked: The term “operably linked” is defined herein as aconfiguration in which a control sequence is appropriately placed at aposition relative to the coding sequence of the DNA sequence such thatthe control sequence directs the expression of a polypeptide.

Coding sequence: When used herein the term “coding sequence” is intendedto cover a nucleotide sequence, which directly specifies the amino acidsequence of its protein product. The boundaries of the coding sequenceare generally determined by an open reading frame, which usually beginswith the ATG start codon. The coding sequence typically includes DNA,cDNA, and recombinant nucleotide sequences.

Expression: In the present context, the term “expression” includes anystep involved in the production of the polypeptide including, but notlimited to, transcription, post-transcriptional modification,translation, post-translational modification, and secretion.

Expression vector: In the present context, the term “expression vector”covers a DNA molecule, linear or circular, that comprises a segmentencoding a polypeptide of the invention, and which is operably linked toadditional segments that provide for its transcription.

Host cell: The term “host cell”, as used herein, includes any cell typewhich is susceptible to transformation with a nucleic acid construct.

The terms “polynucleotide probe”, “hybridization” as well as the variousstringency conditions are defined in the section entitled “PolypeptidesHaving Cellobiohydrolase II Activity”.

Thermostability: The term “thermostability”, as used herein, is measuredas described in Example 2.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides HavingCellobiohydrolase II Activity

In a first embodiment, the present invention relates to polypeptideshaving cellobiohydrolase II activity and where the polypeptidescomprises, preferably consists of, an amino acid sequence which has adegree of identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26.,(i.e., the mature polypeptide) of at least 65%, preferably at least 70%,e.g., at least 75%, more preferably at least 80%, such as at least 85%,even more preferably at least 90%, most preferably at least 95%, e.g.,at least 96%, such as at least 97%, and even most preferably at least98%, such as at least 99% (hereinafter “homologous polypeptides”). In aninteresting embodiment, the amino acid sequence differs by at the mostten amino acids (e.g., by ten amino acids), in particular by at the mostfive amino acids (e.g., by five amino acids), such as by at the mostfour amino acids (e.g., by four amino acids), e.g., by at the most threeamino acids (e.g., by three amino acids) from an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ IDNO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, andSEQ ID NO: 26. In a particular interesting embodiment, the amino acidsequence differs by at the most two amino acids (e.g., by two aminoacids), such as by one amino acid from an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, and SEQID NO: 26.

Preferably, the polypeptides of the present invention comprise an aminoacid sequence selected from the group consisting of SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ IDNO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQID NO: 24, and SEQ ID NO: 26; an allelic variant thereof; or a fragmentthereof that has cellobiohydrolase II activity. In another preferredembodiment, the polypeptide of the present invention consists of anamino acid sequence selected from the group consisting of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO:22, SEQ ID NO: 24, and SEQ ID NO: 26.

The polypeptide of the invention may be a wild-type cellobiohydrolase IIidentified and isolated from a natural source. Such wild-typepolypeptides may be specifically screened for by standard techniquesknown in the art, such as molecular screening as described in Example 1.Furthermore, the polypeptide of the invention may be prepared by the DNAshuffling technique, such as described in Ness et al., 1999, NatureBiotechnology 17: 893-896. Moreover, the polypeptide of the inventionmay be an artificial variant which comprises, preferably consists of, anamino acid sequence that has at least one substitution, deletion and/orinsertion of an amino acid as compared to an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ IDNO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, andSEQ ID NO: 26. Such artificial variants may be constructed by standardtechniques known in the art, such as by site-directed/random mutagenesisof the polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ IDNO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26.In one embodiment of the invention, amino acid changes (in theartificial variant as well as in wild-type polypeptides) are of a minornature, that is conservative amino acid substitutions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of one to about 30 amino acids; small amino orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine, valine andmethionine), aromatic amino acids (phenylalanine, tryptophan andtyrosine), and small amino acids (glycine, alanine, serine andthreonine). Amino acid substitutions which do not generally alter thespecific activity are known in the art and are described, for example,by Neurath and Hill, 1979, In, The Proteins, Academic Press, New York.The most commonly occurring exchanges are Ala/Ser, Val/IIe, Asp/Glu,Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro,Lys/Arg, Asp/Asn, Leu/IIe, Leu/Val, Ala/Glu, and Asp/Gly as well asthese in reverse.

In an interesting embodiment of the invention, the amino acid changesare of such a nature that the physico-chemical properties of thepolypeptides are altered. For example, amino acid changes may beperformed, which improve the thermal stability of the polypeptide, whichalter the substrate specificity, which changes the pH optimum, and thelike.

Preferably, the number of such substitutions, deletions and/orinsertions as compared to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26 is atthe most 10, such as at the most 9, e.g., at the most 8, more preferablyat the most 7, e.g., at the most 6, such as at the most 5, mostpreferably at the most 4, e.g., at the most 3, such as at the most 2, inparticular at the most 1.

The present inventors have isolated nucleotide sequences encodingpolypeptides having cellobiohydrolase II activity from themicroorganisms selected from the group consisting of Chaetomiumthermophilum, Myceliophthora thermophila, Acremonium thermophilum,Thielavia australiensis, Thielavia microspore, Aspergillus tubingensis,Aspergillus tubingensis syn. Aspergillus neotubingensis Frisvad sp.nov., Gloeophyllum trabeum, Meripilus giganteus, Trichophaea saccata,Stilbella annulata, Stilbella annulata and Malbrancheae cinnamomea.

Thus, in a second embodiment, the present invention relates topolypeptides comprising an amino acid sequence which has at least 65%identity with the polypeptide encoded by the cellobiohydrolase IIencoding part of the nucleotide sequence present in an organism selectedfrom the group consisting of Chaetomium thermophilum CGMCC 0859,Myceliophthora thermophila CGMCC 0862, Myceliophthora thermophila CGMCC0862, Acremonium sp. T178-4 CGMCC 0857, Acremonium sp. T178-4,Melanocarpus sp. CGMCC 0861, Thielavia microspora CGMCC 0863,Aspergillus sp. T186-2 CGMCC 0858, Thielavia australiensis CGMCC 0864,Gloeophyllum trabeum ATCC 11.39, Aspergillus tubingensis, CBS 161.79,Trichophaea saccata, CBS 804.70, Stilbella annulata CBS 185.70, andMalbranchea cinnamomea, CBS 115.68. In an interesting embodiment of theinvention, the polypeptide comprises an amino acid sequence which has atleast 70%, e.g., at least 75%, preferably at least 80%, such as at least85%, more preferably at least 90%, most preferably at least 95%, e.g.,at least 96%, such as at least 97%, and even most preferably at least98%, such as at least 99% identity with the polypeptide encoded by thecellobiohydrolase II encoding part of the nucleotide sequence present inan organism selected from the group consisting of Chaetomiumthermophilum CGMCC 0859, Myceliophthora thermophila CGMCC 0862,Myceliophthora thermophila CGMCC 0862, Acremonium sp. T178-4 CGMCC 0857,Acremonium sp. T178-4, Melanocarpus sp. CGMCC 0861, Thielavia microsporaCGMCC 0863, Aspergillus sp. T186-2 CGMCC 0858, Thielavia australiensisCGMCC 0864, Gloeophyllum trabeum ATCC 11.39, Aspergillus tubingensis,CBS 161.79, Trichophaea saccata, CBS 804.70, Stilbella annulata CBS185.70, and Malbranchea cinnamomea, CBS 115.68 (hereinafter “homologouspolypeptides”). In an interesting embodiment, the amino acid sequencediffers by at the most ten amino acids (e.g., by ten amino acids), inparticular by at the most five amino acids (e.g., by five amino acids),such as by at the most four amino acids (e.g., by four amino acids),e.g., by at the most three amino acids (e.g., by three amino acids) fromthe polypeptide encoded by the cellobiohydrolase II encoding part of thenucleotide sequence present in an organism selected from the groupconsisting of Chaetomium thermophilum CGMCC 0859, Myceliophthorathermophila CGMCC 0862, Myceliophthora thermophila CGMCC 0862,Acremonium sp. T178-4 CGMCC 0857, Acremonium sp. T178-4, Melanocarpussp. CGMCC 0861, Thielavia microspora CGMCC 0863, Aspergillus sp. T1862CGMCC 0858, Thielavia australiensis CGMCC 0864, Gloeophyllum trabeumATCC 11.39, Aspergillus tubingensis CBS 161.79, Trichophaea saccata CBS804.70, Stilbella annulata CBS 185.70, and Malbranchea cinnamomea CBS115.68. In a particular interesting embodiment, the amino acid sequencediffers by at the most two amino acids (e.g., by two amino acids), suchas by one amino acid from the polypeptide encoded by thecellobiohydrolase II encoding part of the nucleotide sequence present inan organism selected from the group consisting of Chaetomiumthermophilum CGMCC 0859, Myceliophthora thermophila CGMCC 0862,Myceliophthora thermophila CGMCC 0862, Acremonium sp. T178-4 CGMCC 0857,Acremonium sp. T178-4, Melanocarpus sp. CGMCC 0861, Thielavia microsporaCGMCC 0863, Aspergillus sp. T186-2 CGMCC 0858, Thielavia australiensisCGMCC 0864, Gloeophyllum trabeum ATCC 11.39, Aspergillus tubingensis CBS161.79, Trichophaea saccata CBS 804.70, Stilbella annulata CBS 185.70,and Malbranchea cinnamomea CBS 115.68.

In a third embodiment, the present invention relates to polypeptideshaving cellobiohydrolase II activity which are encoded by nucleotidesequences which hybridize under very low stringency conditions,preferably under low stringency conditions, more preferably under mediumstringency conditions, more preferably under medium-high stringencyconditions, even more preferably under high stringency conditions, andmost preferably under very high stringency conditions with apolynucleotide probe selected from the group consisting of thecomplementary strand of the nucleotides selected from the groupconsisting of:

nucleotides 63 to 1493 of SEQ ID NO: 1,

nucleotides 1 to 246 of SEQ ID NO: 3,

nucleotides 1 to 417 of SEQ ID NO: 5,

nucleotides 1 to 306 of SEQ ID NO: 7,

nucleotides 1 to 432 of SEQ ID NO: 9,

nucleotides 1 to 297 of SEQ ID NO: 11,

nucleotides 1 to 420 of SEQ ID NO: 13,

nucleotides 1 to 330 of SEQ ID NO: 15,

nucleotides 1 to 1221 of SEQ ID NO: 15,

nucleotides 1 to 429 of SEQ ID NO: 17,

nucleotides 1 to 213 of SEQ ID NO: 19,

nucleotides 43 to 701 of SEQ ID NO: 21,

nucleotides 21 to 1394 of SEQ ID NO: 23, and

nucleotides 41 to 1210 of SEQ ID NO: 25.

In another embodiment, the present invention relates to polypeptideshaving cellobiohydrolase II activity which are encoded by thecellobiohydrolase II encoding part of the nucleotide sequence present ina microorganism selected from the group consisting of:

a microorganism belonging to the family Chaetomiaceae, preferably to thegenus Chaetomium, more preferably to the species Chaetomiumthermophilum,

a microorganism belonging to the genus Myceliophthora, preferably to thespecies Myceliophthora thermophila,

a microorganism belonging to the species Acremonium thermophilum,

a microorganism belonging to the family Chaetomiaceae, preferably to thegenus Thielavia, preferably to the species Thielavia australiensis,

a microorganism belonging to the genus Aspergillus, preferably belongingto the black Aspergilli,

a microorganism belonging to the family Chaetomiaceae, preferably to thegenus Thielavia, preferably to the species Thielavia microspore,

a microorganism belonging to the genus Aspergillus, preferably belongingto the black Aspergilli, more preferably to the species Aspergillustubingensis, and most preferably to the species A. neotubingensisFrisvad sp. nov.

a microorganism belonging to the Polyporales, preferably belonging tothe family Fomitopsidacea, more preferably belonging to the genusGloeophyllum, most preferably to the species Gloeophyllum trabeum,

a microorganism belonging to the Hymenochaetales, preferably belongingto the family Rigidiporaceae, preferably belonging to the genusMeripilus, more preferably to the species Meripilus giganteus,

a microorganism belonging to the Pezizomycotina, preferably belonging toPezizales, preferably belonging to the family Pyronemataceae or thefamily Sarcosomataceae, more preferably belonging to the genusTrichophaea or the genus Pseudoplectania, most preferably Trichophaeasaccata,

a microorganism belonging to the species Stilbella annulata, and

a microorganism belonging to the species Malbrancheae cinnamomea.

A nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ IDNO: 21, and SEQ ID NO: 23, and SEQ ID NO: 25 or a subsequence thereof,as well as an amino acid sequence selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26, or a fragmentthereof, may be used to design a polynucleotide probe to identify andclone DNA encoding polypeptides having cellobiohydrolase 11 activityfrom strains of different genera or species according to methods wellknown in the art. In particular, such probes can be used forhybridization with the genomic or cDNA of the genus or species ofinterest, following standard Southern blotting procedures, in order toidentify and isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, preferably at least 25, more preferably at least 35 nucleotides inlength, such as at least 70 nucleotides in length. It is, however,preferred that the polynucleotide probe is at least 100 nucleotides inlength. For example, the polynucleotide probe may be at least 200nucleotides in length, at least 300 nucleotides in length, at least 400nucleotides in length or at least 500 nucleotides in length. Even longerprobes may be used, e.g., polynucleotide probes which are at least 600nucleotides in length, at least 700 nucleotides in length, at least 800nucleotides in length, or at least 900 nucleotides in length. Both DNAand RNA probes can be used. The probes are typically labeled fordetecting the corresponding gene (for example, with ³²P, ³H, ³⁵S,biotin, or avidin).

Thus, a genomic DNA or cDNA library prepared from such other organismsmay be screened for DNA which hybridizes with the probes described aboveand which encodes a polypeptide having cellobiohydrolase II activity.Genomic or other DNA from such other organisms may be separated byagarose or polyacrylamide gel electrophoresis, or other separationtechniques. DNA from the libraries or the separated DNA may betransferred to, and immobilized, on nitrocellulose or other suitablecarrier materials. In order to identify a clone or DNA which ishomologous with one of the sequence shown in SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13,SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO:23, and SEQ ID NO: 25, the carrier material with the immobilized DNA isused in a Southern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled polynucleotide probe whichhybridizes to any of the nucleotide sequences shown in SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21,SEQ ID NO: 23, and SEQ ID NO: 25 under very low to very high stringencyconditions. Molecules to which the polynucleotide probe hybridizes underthese conditions may be detected using X-ray film or by any other methodknown in the art. Whenever the term “polynucleotide probe” is used inthe present context, it is to be understood that such a probe containsat least 15 nucleotides.

In an interesting embodiment, the polynucleotide probe is thecomplementary strand of the nucleotides selected from the groupconsisting of:

nucleotides 63 to 1493 of SEQ ID NO: 1,

nucleotides 1 to 246 of SEQ ID NO: 3,

nucleotides 1 to 1272 of SEQ ID NO: 3,

nucleotides 1 to 417 of SEQ ID NO: 5,

nucleotides 1 to 306 of SEQ ID NO: 7,

nucleotides 1 to 432 of SEQ ID NO: 9,

nucleotides 1 to 297 of SEQ ID NO: 11,

nucleotides 1 to 420 of SEQ ID NO: 13,

nucleotides 1 to 330 of SEQ ID NO: 15,

nucleotides 1 to 1221 of SEQ ID NO: 15,

nucleotides 1 to 429 of SEQ ID NO: 17,

nucleotides 1 to 213 of SEQ ID NO: 19,

nucleotides 43 to 701 of SEQ ID NO: 21,

nucleotides 21 to 1394 of SEQ ID NO: 23, and

nucleotides 41 to 1210 of SEQ ID NO: 25.

In another interesting embodiment, the polynucleotide probe is thecomplementary strand of the nucleotide sequence which encodes apolypeptide selected from the group consisting of SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ IDNO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQID NO: 24, and SEQ ID NO: 26. In a further interesting embodiment, thepolynucleotide probe is the complementary strand of a nucleotidesequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ IDNO: 23 and SEQ ID NO: 25.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 1.0% SDS, 5×Denhardt's solution, 100microg/ml sheared and denatured salmon sperm DNA, following standardSouthern blotting procedures. Preferably, the long probes of at least100 nucleotides do not contain more than 1000 nucleotides. For longprobes of at least 100 nucleotides in length, the carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.1% SDS at42° C. (very low stringency), preferably washed three times each for 15minutes using 0.5×SSC, 0.1% SDS at 42° C. (low stringency), morepreferably washed three times each for 15 minutes using 0.2×SSC, 0.1%SDS at 42° C. (medium stringency), even more preferably washed threetimes each for 15 minutes using 0.2×SSC, 0.1% SDS at 55° C. (medium-highstringency), most preferably washed three times each for 15 minutesusing 0.1×SSC, 0.1% SDS at 60° C. (high stringency), in particularwashed three times each for 15 minutes using 0.1×SSC, 0.1% SDS at 68° C.(very high stringency).

Although not particularly preferred, it is contemplated that shorterprobes, e.g., probes which are from about 15 to 99 nucleotides inlength, such as from about 15 to about 70 nucleotides in length, may bealso be used. For such short probes, stringency conditions are definedas prehybridization, hybridization, and washing post-hybridization at 5°C. to 10° C. below the calculated T_(m) using the calculation accordingto Bolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures.

For short probes which are about 15 nucleotides to 99 nucleotides inlength, the carrier material is washed once in 6×SCC plus 0.1% SDS for15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C.below the calculated T_(m).

Sources for Polypeptides Having Cellobiohydrolase II Activity

A polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein shall mean that the polypeptideencoded by the nucleotide sequence is produced by a cell in which thenucleotide sequence is naturally present or into which the nucleotidesequence has been inserted. In a preferred embodiment, the polypeptideis secreted extracellularly.

A polypeptide of the present invention may be a bacterial polypeptide.For example, the polypeptide may be a gram positive bacterialpolypeptide such as a Bacillus polypeptide, e.g., a Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus coagulans, Bacillus lautus, Bacillus lentus,Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, or Bacillus thuringiensispolypeptide; or a Streptomyces polypeptide, e.g., a Streptomyceslividans or Streptomyces murinus polypeptide; or a gram negativebacterial polypeptide, e.g., an E. coli or a Pseudomonas sp.polypeptide.

A polypeptide of the present invention may be a fungal polypeptide, andpreferably a yeast polypeptide such as a Candida, Kluyveromyces,Neocallimastix, Pichia, Piromyces, Saccharomyces, Schizosaccharomyces,or Yarrowia polypeptide; or more preferably a filamentous fungalpolypeptide such as an Acremonium, Aspergillus, Chaetomium, Chaetomium,Gloeophyllum, Malbrancheae, Melanocarpus, Meripilus, Myceliophthora,Stilbella, Thielavia, or Trichophaea polypeptide.

In an interesting embodiment, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensisor Saccharomyces oviformis polypeptide.

In a preferred embodiment, the polypeptide is a Chaetomium thermophilum,Myceliophthora thermophila, Acremonium thermophilum, Thielaviaaustraliensis, Aspergilli, Thielavia microspore, Aspergillustubingensis, Gloeophyllum trabeum, Meripilus giganteus, Trichophaeasaccata, Stilbella annulata, or Malbrancheae cinnamomea polypeptide

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,water, plants, animals, etc.) using the above-mentioned probes.Techniques for isolating microorganisms from natural habitats are wellknown in the art. The nucleotide sequence may then be derived bysimilarly screening a genomic or cDNA library of another microorganism.Once a nucleotide sequence encoding a polypeptide has been detected withthe probe(s), the sequence may be isolated or cloned by utilizingtechniques which are known to those of ordinary skill in the art (see,e.g., Sambrook, Fritsch, and Maniatus, 1989, Molecular Cloning, ALaboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).

Polypeptides encoded by nucleotide sequences of the present inventionalso include fused polypeptides or cleavable fusion polypeptides inwhich another polypeptide is fused at the N-terminus or the C-terminusof the polypeptide or fragment thereof. A fused polypeptide is producedby fusing a nucleotide sequence (or a portion thereof) encoding anotherpolypeptide to a nucleotide sequence (or a portion thereof of thepresent invention. Techniques for producing fusion polypeptides areknown in the art, and include ligating the coding sequences encoding thepolypeptides so that they are in frame and that expression of the fusedpolypeptide is under control of the same promoter(s) and terminator.

Polynucleotides and Nucleotide Sequences

The present invention also relates to polynucleotides having anucleotide sequence which encodes for a polypeptide of the invention. Inparticular, the present invention relates to polynucleotides consistingof a nucleotide sequence which encodes for a polypeptide of theinvention. In a preferred embodiment, the nucleotide sequence isselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ IDNO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 andSEQ ID NO: 25. The present invention also encompasses polynucleotidescomprising, preferably consisting of, nucleotide sequences which encodea polypeptide consisting of an amino acid sequence selected from thegroup consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ IDNO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26,which differ from a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 and SEQ ID NO: 25 byvirtue of the degeneracy of the genetic code.

The present invention also relates to polynucleotides comprising,preferably consisting of, a subsequence of a nucleotide sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ IDNO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 andSEQ ID NO: 25 which encode fragments of an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, and SEQID NO: 26, that have cellobiohydrolase II activity. A subsequence of anucleotide sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO: 23 and SEQ ID NO: 25 is a nucleotide sequence encompassedby a sequence selected from the group consisting of SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ IDNO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQID NO: 23 and SEQ ID NO: 25 except that one or more nucleotides from the5′ and/or 3′ end have been deleted.

The present invention also relates to polynucleotides having, preferablyconsisting of, a modified nucleotide sequence which comprises at leastone modification in the mature polypeptide coding sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 and SEQ ID NO: 25,and where the modified nucleotide sequence encodes a polypeptide whichconsists of an amino acid sequence selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 26.

The techniques used to isolate or clone a nucleotide sequence encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thenucleotide sequences of the present invention from such genomic DNA canbe effected, e.g., by using the well known polymerase chain reaction(PCR) or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other amplification procedures such as ligase chain reaction (LCR),ligated activated transcription (LAT) and nucleotide sequence-basedamplification (NASBA) may be used. The nucleotide sequence may be clonedfrom a strain selected from a strain belonging to a genus selected fromthe group consisting of Chaetomium, Myceliophthora, Melanocarpus,Acremonium, Thielavia, Aspergillus, Gloeophyllum, Meripilus,Trichophaea, Stilbella and Malbrancheae, or another or related organismand thus, for example, may be an allelic or species variant of thepolypeptide encoding region of the nucleotide sequence.

The nucleotide sequence may be obtained by standard cloning proceduresused in genetic engineering to relocate the nucleotide sequence from itsnatural location to a different site where it will be reproduced. Thecloning procedures may involve excision and isolation of a desiredfragment comprising the nucleotide sequence encoding the polypeptide,insertion of the fragment into a vector molecule, and incorporation ofthe recombinant vector into a host cell where multiple copies or clonesof the nucleotide sequence will be replicated. The nucleotide sequencemay be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or anycombinations thereof.

The present invention also relates to a polynucleotide comprising,preferably consisting of, a nucleotide sequence which has a degree ofidentity with a nucleotide sequence selected from the group consistingof:

nucleotides 63 to 1493 of SEQ ID NO: 1,

nucleotides 1 to 246 of SEQ ID NO: 3,

nucleotides 1 to 1272 of SEQ ID NO: 3,

nucleotides 1 to 417 of SEQ ID NO: 5,

nucleotides 1 to 306 of SEQ ID NO: 7,

nucleotides 1 to 432 of SEQ ID NO: 9,

nucleotides 1 to 297 of SEQ ID NO: 11,

nucleotides 1 to 420 of SEQ ID NO: 13,

nucleotides 1 to 330 of SEQ ID NO: 15,

nucleotides 1 to 1221 of SEQ ID NO: 15,

nucleotides 1 to 429 of SEQ ID NO: 17,

nucleotides 1 to 213 of SEQ ID NO: 19,

nucleotides 43 to 701 of SEQ ID NO: 21,

nucleotides 21 to 1394 of SEQ ID NO: 23, and nucleotides 41 to 1210 ofSEQ ID NO: 25

of at least 70% identity, such as at least 75% identity; preferably, thenucleotide sequence has at least 80% identity, e.g., at least 85%identity, such as at least 90% identity, more preferably at least 95%identity, such as at least 96% identity, e.g., at least 97% identity,even more preferably at least 98% identity, such as at least 99%.Preferably, the nucleotide sequence encodes a polypeptide havingcellobiohydrolase II activity. The degree of identity between twonucleotide sequences is determined as described previously (see thesection entitled “Definitions”).

Modification of a nucleotide sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of a polypeptide,which comprises an amino acid sequence that has at least onesubstitution, deletion and/or insertion as compared to an amino acidsequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ IDNO: 26 and SEQ ID NO: 28. These artificial variants may differ in someengineered way from the polypeptide isolated from its native source,e.g., variants that differ in specific activity, thermostability or pHoptimum.

It will be apparent to those skilled in the art that such modificationscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by the nucleotide sequence ofthe invention, and therefore preferably not subject to modification,such as substitution, may be identified according to procedures known inthe art, such as site-directed mutagenesis or alanine-scanningmutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, mutations are introduced at everypositively charged residue in the molecule, and the resultant mutantmolecules are tested for cellobiohydrolase II activity to identify aminoacid residues that are critical to the activity of the molecule. Sitesof substrate-enzyme interaction can also be determined by analysis ofthe three-dimensional structure as determined by such techniques asnuclear magnetic resonance analysis, crystallography or photoaffinitylabelling (see, e.g., de Vos et al., 1992, Science 255: 306312; Smith etal., 1992, Journal of Molecular Biology 224: 899904; Wlodaver et al.,1992, FEBS Letters 309: 59-64).

Moreover, a nucleotide sequence encoding a polypeptide of the presentinvention may be modified by introduction of nucleotide substitutionswhich do not give rise to another amino acid sequence of the polypeptideencoded by the nucleotide sequence, but which correspond to the codonusage of the host organism intended for production of the enzyme.

The introduction of a mutation into the nucleotide sequence to exchangeone nucleotide for another nucleotide may be accomplished bysite-directed mutagenesis using any of the methods known in the art.Particularly useful is the procedure, which utilizes a supercoiled,double stranded DNA vector with an insert of interest and two syntheticprimers containing the desired mutation. The oligonucleotide primers,each complementary to opposite strands of the vector, extend duringtemperature cycling by means of Pfu DNA polymerase. On incorporation ofthe primers, a mutated plasmid containing staggered nicks is generated.Following temperature cycling, the product is treated with DpnI which isspecific for methylated and hemimethylated DNA to digest the parentalDNA template and to select for mutation-containing synthesized DNA.Other procedures known in the art may also be used. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

The present invention also relates to a polynucleotide comprising,preferably consisting of, a nucleotide sequence which encodes apolypeptide having cellobiohydrolase II activity, and which hybridizesunder very low stringency conditions, preferably under low stringencyconditions, more preferably under medium stringency conditions, morepreferably under medium-high stringency conditions, even more preferablyunder high stringency conditions, and most preferably under very highstringency conditions with a polynucleotide probe selected from thegroup consisting of

(i) the complementary strand of the nucleotides selected from the groupconsisting of:

-   -   nucleotides 63 to 1493 of SEQ ID NO: 1,    -   nucleotides 1 to 246 of SEQ ID NO: 3,    -   nucleotides 1 to 1272 of SEQ ID NO: 3,    -   nucleotides 1 to 417 of SEQ ID NO: 5,    -   nucleotides 1 to 306 of SEQ ID NO: 7,    -   nucleotides 1 to 432 of SEQ ID NO: 9,    -   nucleotides 1 to 297 of SEQ ID NO: 11,    -   nucleotides 1 to 420 of SEQ ID NO: 13,    -   nucleotides 1 to 330 of SEQ ID NO: 15,    -   nucleotides 1 to 1221 of SEQ ID NO: 15,    -   nucleotides 1 to 429 of SEQ ID NO: 17,    -   nucleotides 1 to 213 of SEQ ID NO: 19,    -   nucleotides 43 to 701 of SEQ ID NO: 21,    -   nucleotides 21 to 1394 of SEQ ID NO: 23, and    -   nucleotides 41 to 1210 of SEQ ID NO: 25.

As will be understood, details and particulars concerning hybridizationof the nucleotide sequences will be the same or analogous to thehybridization aspects discussed in the section entitled “PolypeptidesHaving Cellobiohydrolase II Activity” herein.

DNA Recombination (Shuffling)

The nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 and SEQ IDNO: 25 may be used in a DNA recombination (or shuffling) process. Thenew polynucleotide sequences obtained in such a process may encode newpolypeptides having cellobiase activity with improved properties, suchas improved stability (storage stability, thermostability), improvedspecific activity, improved pH-optimum, and/or improved tolerancetowards specific compounds.

Shuffling between two or more homologous input polynucleotides(starting-point polynucleotides) involves fragmenting thepolynucleotides and recombining the fragments, to obtain outputpolynucleotides (i.e., polynucleotides that have been subjected to ashuffling cycle) wherein a number of nucleotide fragments are exchangedin comparison to the input polynucleotides.

DNA recombination or shuffling may be a (partially) random process inwhich a library of chimeric genes is generated from two or more startinggenes. A number of known formats can be used to carry out this shufflingor recombination process.

The process may involve random fragmentation of parental DNA followed byreassembly by PCR to new full-length genes, e.g., as presented in U.S.Pat. Nos. 5,605,793, 5,811,238, 5,830,721, and 6,117,679. In-vitrorecombination of genes may be carried out, e.g., as described in U.S.Pat. Nos. 6,159,687, 6,159,688, 5,965,408, and 6,153,510 and WO98/41623. The recombination process may take place in vivo in a livingcell, e.g., as described in WO 97/07205 and WO 98/28416.

The parental DNA may be fragmented by DNA'se I treatment or byrestriction endonuclease digests as described by Kikuchi et al. (2000,Gene 236: 159-167). Shuffling of two parents may be done by shufflingsingle stranded parental DNA of the two parents as described in Kikuchiet al., (2000, Gene 243: 133-137).

A particular method of shuffling is to follow the methods described inCrameri et al, 1998, Nature 391: 288-291 and Ness et al., NatureBiotechnology 17: 893-896. Another format would be the methods describedin U.S. Pat. No. 6,159,687: Examples 1 and 2.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga nucleotide sequence of the present invention operably linked to one ormore control sequences that direct the expression of the coding sequencein a suitable host cell under conditions compatible with the controlsequences.

A nucleotide sequence encoding a polypeptide of the present inventionmay be manipulated in a variety of ways to provide for expression of thepolypeptide. Manipulation of the nucleotide sequence prior to itsinsertion into a vector may be desirable or necessary depending on theexpression vector. The techniques for modifying nucleotide sequencesutilizing recombinant DNA methods are well known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence which is recognized by a host cell for expression ofthe nucleotide sequence. The promoter sequence contains transcriptionalcontrol sequences, which mediate the expression of the polypeptide. Thepromoter may be any nucleotide sequence which shows transcriptionalactivity in the host cell of choice including mutant, truncated, andhybrid promoters, and may be obtained from genes encoding extracellularor intracellular polypeptides either homologous or heterologous to thehost cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, andFusarium oxysporum trypsin-like protease (WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C(CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and which,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencewhich is functional in the host cell of choice may be used in thepresent invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GALL systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleotide sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising the nucleic acid construct of the invention. The variousnucleotide and control sequences described above may be joined togetherto produce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleotide sequence encoding the polypeptide at such sites.Alternatively, the nucleotide sequence of the present invention may beexpressed by inserting the nucleotide sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of the nucleotide sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.

The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers which confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),trpC (anthranilate synthase), as well as equivalents thereof.

Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits stable integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome.

For integration into the host cell genome, the vector may rely on thenucleotide sequence encoding the polypeptide or any other element of thevector for stable integration of the vector into the genome byhomologous or nonhomologous recombination. Alternatively, the vector maycontain additional nucleotide sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleotide sequences enable the vector to be integrated intothe host cell genome at a precise location(s) in the chromosome(s). Toincrease the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleotides, such as 100 to 1,500 base pairs, preferably 400 to 1,500base pairs, and most preferably 800 to 1,500 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleotide sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are theorigins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1permitting replication in Bacillus. Examples of origins of replicationfor use in a yeast host cell are the 2 micron origin of replication,ARS1, ARS4, the combination of ARS1 and CEN3, and the combination ofARS4 and CEN6. The origin of replication may be one having a mutationwhich makes its functioning temperature-sensitive in the host cell (see,e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA75: 1433).

More than one copy of a nucleotide sequence of the present invention maybe inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleotide sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleotide sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleotide sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant a host cell comprisingthe nucleic acid construct of the invention, which are advantageouslyused in the recombinant production of the polypeptides. A vectorcomprising a nucleotide sequence of the present invention is introducedinto a host cell so that the vector is maintained as a chromosomalintegrant or as a self-replicating extra-chromosomal vector as describedearlier.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote.

Useful unicellular cells are bacterial cells such as gram positivebacteria including, but not limited to, a Bacillus cell, e.g., Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus clausii, Bacillus coagulans, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or aStreptomyces cell, e.g., Streptomyces lividans or Streptomyces murinus,or gram negative bacteria such as E. coli and Pseudomonas sp. In apreferred embodiment, the bacterial host cell is a Bacillus lentus,Bacillus licheniformis, Bacillus stearothermophilus, or Bacillussubtilis cell. In another preferred embodiment, the Bacillus cell is analkalophilic Bacillus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169; 5771-5278).

The host cell may be a eukaryote, such as a mammalian, insect, plant, orfungal cell.

In a preferred embodiment, the host cell is a fungal cell. “Fungi” asused herein includes the phyla Ascomycota, Basidiomycota,Chytridiomycota, and Zygomycota (as defined by Hawksworth et al, In,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK) as well as the Oomycota(as cited in Hawksworth et al., 1995, supra, page 171) and allmitosporic fungi (Hawksworth et al., 1995, supra).

In a more preferred embodiment, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred embodiment, the yeast host cell is a Candida,Aschbyii, Hansenula, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia cell.

In a most preferred embodiment, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensisor Saccharomyces oviformis cell. In another most preferred embodiment,the yeast host cell is a Kluyveromyces lactis cell. In another mostpreferred embodiment, the yeast host cell is a Yarrowia lipolytica cell.

In another more preferred embodiment, the fungal host cell is afilamentous fungal cell. “Filamentous fungi” include all filamentousforms of the subdivision Eumycota and Oomycota (as defined by Hawksworthet al., 1995, supra). The filamentous fungi are characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred embodiment, the filamentous fungal host cellis a cell of a species of, but not limited to, Acremonium, Aspergillus,Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium,Thielavia, Tolypocladium, or Trichoderma.

In a most preferred embodiment, the filamentous fungal host cell is anAspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. Inanother most preferred embodiment, the filamentous fungal host cell is aFusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, or Fusarium venenatum cell. In an even mostpreferred embodiment, the filamentous fungal parent cell is a Fusariumvenenatum (Nirenberg sp. nov.) cell. In another most preferredembodiment, the filamentous fungal host cell is a Humicola insolens,Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson and Simon, editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, 194: 182-187, Academic Press,Inc., New York; Ito et al., 1983, Journal of Bacteriology 153: 163; andHinnen et al, 1978, Proceedings of the National Academy of Sciences USA75: 1920.

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating astrain, which in its wild-type form is capable of producing thepolypeptide; and (b) recovering the polypeptide. Preferably, the strainis selected from a species within a genus comprised in the groupconsisting of Acremonium, Aspergillus, Chaetomium, Chaetomium,Gloeophyllum, Malbrancheae, Melanocarpus, Meripilus, Myceliophthora,Stilbella, Thielavia, or Trichophaea; more preferably the strain isselected from the group consisting of Chaetomium thermophilum,Myceliophthora thermophila, Thielavia australiensis, Thielaviamicrospore, Aspergillus sp., the black Aspergilli, Aspergillustubingensis syn. A. neotubingensis Frisvad sp. nov., Gloeophyllumtrabeum, Meripilus giganteus, Trichophaea saccata, Stilbella annulata,and Malbrancheae cinnamomea.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

The present invention also relates to methods for in-situ production ofa polypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) contacting the polypeptide with a desired substrate, such as acellulosic substrate, without prior recovery of the polypeptide. Theterm “in-situ production” is intended to mean that the polypeptide isproduced directly in the locus in which it is intended to be used, suchas in a fermentation process for production of ethanol.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered by methods known in the art.For example, the polypeptide may be recovered from the nutrient mediumby conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, Janson and Ryden, editors, VCH Publishers, NewYork, 1989).

Plants

The present invention also relates to a transgenic plant, plant part, orplant cell which has been transformed with a nucleotide sequenceencoding a polypeptide having cellobiohydrolase II activity of thepresent invention so as to express and produce the polypeptide inrecoverable quantities. The polypeptide may be recovered from the plantor plant part. Alternatively, the plant or plant part containing therecombinant polypeptide may be used as such for improving the quality ofa food or feed, e.g., improving nutritional value, palatability, andrheological properties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, millets, and maize (corn).

Examples of dicot plants are tobacco, lupins, potato, sugar beet,legumes, such as pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape, canola, and the closelyrelated model organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers. Also specific plant tissues, such as chloroplast, apoplast,mitochondria, vacuole, peroxisomes, and cytoplasm are considered to be aplant part. Furthermore, any plant cell, whatever the tissue origin, isconsidered to be a plant part.

Also included within the scope of the present invention are the progeny(clonal or seed) of such plants, plant parts and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. Briefly, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome and propagating theresulting modified plant or plant cell into a transgenic plant or plantcell.

Conveniently, the expression construct is a nucleic acid construct whichcomprises a nucleotide sequence encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleotide sequence in the plant or plant part ofchoice. Furthermore, the expression construct may comprise a selectablemarker useful for identifying host cells into which the expressionconstruct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague etal., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV promoter may be used (Francket al., 1980, Cell 21: 285-294). Organ-specific promoters may be, forexample, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu et al.,1993, Plant Molecular Biology 22: 573-588).

A promoter enhancer element may also be used to achieve higherexpression of the enzyme in the plant. For instance, the promoterenhancer element may be an intron which is placed between the promoterand the nucleotide sequence encoding a polypeptide of the presentinvention. For instance, Xu et al., 1993, supra disclose the use of thefirst intron of the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38).However it can also be used for transforming monocots, although othertransformation methods are generally preferred for these plants.Presently, the method of choice for generating transgenic monocots isparticle bombardment (microscopic gold or tungsten particles coated withthe transforming DNA) of embryonic calli or developing embryos(Christou, 1992, Plant Journal 2: 275-281; Shimamoto, 1994, CurrentOpinion Biotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

Following transformation, the transformants having incorporated thereinthe expression construct are selected and regenerated into whole plantsaccording to methods well-known in the art.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a nucleotide sequenceencoding a polypeptide having cellobiohydrolase II activity of thepresent invention under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide.

The present invention also relates to methods for in-situ production ofa polypeptide of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a nucleotide sequenceencoding a polypeptide having cellobiohydrolase II activity of thepresent invention under conditions conducive for production of thepolypeptide; and (b) contacting the polypeptide with a desiredsubstrate, such as a cellulosic substrate, without prior recovery of thepolypeptide.

Compositions

In a still further aspect, the present invention relates to compositionscomprising a polypeptide of the present invention.

The composition may comprise a polypeptide of the invention as the majorenzymatic component, e.g., a mono-component composition. Alternatively,the composition may comprise multiple enzymatic activities, such as anaminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase,cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase,invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme,peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, proteolyticenzyme, ribonuclease, transglutaminase, or xylanase.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Forinstance, the polypeptide composition may be in the form of a granulateor a microgranulate. The polypeptide to be included in the compositionmay be stabilized in accordance with methods known in the art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Detergent Compositions

The polypeptide of the invention may be added to and thus become acomponent of a detergent composition.

The detergent composition of the invention may for example be formulatedas a hand or machine laundry detergent composition including a laundryadditive composition suitable for pre-treatment of stained fabrics and arinse added fabric softener composition, or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dishwashing operations.

In a specific aspect, the invention provides a detergent additivecomprising the polypeptide of the invention. The detergent additive aswell as the detergent composition may comprise one or more other enzymessuch as a protease, a lipase, a cutinase, an amylase, a carbohydrase, acellulase, a pectinase, a mannanase, an arabinase, a galactanase, axylanase, an oxidase, e.g., a laccase, and/or a peroxidase.

In general the properties of the chosen enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Proteases: Suitable proteases include those of animal, vegetable ormicrobial origin. Microbial origin is preferred. Chemically modified orprotein engineered mutants are included. The protease may be a serineprotease or a metallo protease, preferably an alkaline microbialprotease or a trypsin-like protease. Examples of alkaline proteases aresubtilisins, especially those derived from Bacillus, e.g., subtilisinNovo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 andsubtilisin 168 (described in WO 89/06279). Examples of trypsin-likeproteases are trypsin (e.g., of porcine or bovine origin) and theFusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729,WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants withsubstitutions in one or more of the following positions: 27, 36, 57, 76,87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and274.

Lipases: Suitable lipases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful lipases include lipases from Humicola (synonym Thermomyces),e.g., from H. lanuginosa (T. lanuginosus) as described in EP 258 068 andEP 305 216 or from H. insolens as described in WO 96/13580, aPseudomonas lipase, e.g., from P. alcaligenes or P. pseudoalcaligenes(EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P.fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g.,from B. subtilis (Dartois et al., 1993, Biochemica et Biophysica Acta1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO91/16422).

Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202.

Amylases: Suitable amylases (alpha and/or beta) include those ofbacterial or fungal origin. Chemically modified or protein engineeredmutants are included. Amylases include, for example, alpha-amylasesobtained from Bacillus, e.g., a special strain of B. licheniformis,described in more detail in GB 1,296,839.

Examples of useful amylases are the variants described in WO 94/02597,WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants withsubstitutions in one or more of the following positions: 15, 23, 105,106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243,264, 304, 305, 391, 408, and 444.

Cellulases: Suitable cellulases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Suitable cellulases include cellulases from the genera Bacillus,Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungalcellulases produced from Humicola insolens, Myceliophthora thermophilaand Fusarium oxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263,5,691,178, and 5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving colour care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, andWO 98/08940. Other examples are cellulase variants such as thosedescribed in WO 94/07998, EP 0 531 315, U.S. Pat. Nos. 5,457,046,5,686,593, and 5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those ofplant, bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Examples of useful peroxidases includeperoxidases from Coprinus, e.g., from C. cinereus, and variants thereofas those described in WO 93/24618, WO 95/10602, and WO 98/15257.

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e., a separate additive or a combined additive, canbe formulated, e.g., as a granulate, a liquid, a slurry, etc. Preferreddetergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition of the invention may be in any convenientform, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. Aliquid detergent may be aqueous, typically containing up to 70% waterand 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of from0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g., SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxybenzenesulfonate. Alternatively, the bleaching system maycomprise peroxyacids of e.g., the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in, e.g., WO 92/19709and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

It is at present contemplated that in the detergent compositions anyenzyme, in particular the polypeptide of the invention, may be added inan amount corresponding to 0.01-100 mg of enzyme protein per liter ofwash liquor, preferably 0.05-5 mg of enzyme protein per liter of washliquor, in particular 0.1-1 mg of enzyme protein per liter of washliquor.

The polypeptide of the invention may additionally be incorporated in thedetergent formulations disclosed in WO 97/07202 which is herebyincorporated as reference.

Production of Ethanol from Biomass

The present invention also relates to methods for producing ethanol frombiomass, such as cellulosic materials, comprising contacting the biomasswith the polypeptides of the invention. Ethanol may subsequently berecovered. The polypeptides of the invention may be produced “in-situ”,i.e., as part of, or directly in an ethanol production process, bycultivating a host cell or a strain, which in its wild-type form iscapable of producing the polypeptides, under conditions conducive forproduction of the polypeptides.

Ethanol can be produced by enzymatic degradation of biomass andconversion of the released polysaccharides to ethanol. This kind ofethanol is often referred to as bioethanol or biofuel. It can be used asa fuel additive or extender in blends of from less than 1% and up to100% (a fuel substitute). In some countries, such as Brazil, ethanol issubstituting gasoline to a very large extent.

The predominant polysaccharide in the primary cell wall of biomass iscellulose, the second most abundant is hemi-cellulose, and the third ispectin. The secondary cell wall, produced after the cell has stoppedgrowing, also contains polysaccharides and is strengthened throughpolymeric lignin covalently cross-linked to hemicellulose. Cellulose isa homopolymer of anhydrocellobiose and thus a linearbeta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which helps stabilize the cell wall matrix.

Three major classes of cellulase enzymes are used to breakdown biomass:

-   -   The “endo-1,4-beta-glucanases” or        1,4-beta-D-glucan-4-glucanohydrolases (EC 3.2.1.4), which act        randomly on soluble and insoluble 1,4-beta-glucan substrates.    -   The “exo-1,4-beta-D-glucanases” including both the        1,4-beta-D-glucan glucohydrolases (EC 3.2.1.74), which liberate        D-glucose from 1,4-beta-D-glucans and hydrolyze D-cellobiose        slowly, and 1,4-beta-D-glucan cellobiohydrolase (EC 3.2.1.91),        also referred to as cellobiohydrolase I and II, which liberates        D-cellobiose from 1,4-beta-glucans.    -   The “beta-D-glucosidases” or beta-D-glucoside glucohydrolases        (EC 3.2.1.21), which act to release D-glucose units from        cellobiose and soluble cellodextrins, as well as an array of        glycosides.

These three classes of enzymes work together synergistically in acomplex interplay that results in efficient decrystallization andhydrolysis of native cellulose from biomass to yield the reducing sugarswhich are converted to ethanol by fermentation.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Example 1 Molecular Screening of Cellobiohydrase II from ThermophilicFungi

The fungal strains were grown in 80 ml liquid media (2.5% Avicel, 0.5%Glucose, 0.14% (NH₄)₂SO₄) in 500 ml Erlenmeyer flasks. The flasks wereincubated for 72 hours at 45° C. on a rotary shaker at 165 rpm. Myceliumwas harvested by centrifugation at 7000 rpm for 30 minutes and stored at−80° C. before use for RNA extraction.

Total RNA was extracted from 100 mg mycelium of each strain using theRNeasy Mini Kit (QIAGEN, Cat. No. 74904).

Degenerate primers were designed based on alignment of already knownCBHII protein sequences. The following primers were designed (see alsoSEQ ID NOS: 27 to 32).

SEQ ID NO: 27 CBHII 1S: 5′ TGG GGN CA(A/G) TG(T/C) GGN GG 3′ SEQ ID NO:28 CBHII 2S: 5′ TGG (T/C)TN GGN TGG CCN GC 3′ SEQ ID NO: 29 CBHII 2AS:5′ GCN GGC CAN CCN A(A/G)C CA 3′ (reverse) SEQ ID NO: 30 CBHII 3AS:5′ TT(A/G) CAC CA(A/G) TCN CCC CA 3′ (reverse) SEQ ID NO: 31 CBHII 4AS:5′ GG(T/C) TTN ACC CAN AC(A/G) AA 3′ (reverse) SEQ ID NO: 32 CBHII 5AS:5′ AA(A/G) TAN GC(T/C) TG(A/G) AAC CA 3′ (reverse)

The 3′ RACE system (GIBCO., Cat. No. 18373-019) were used to synthesizecDNA from total RNA. About 5 micrograms total RNA was used as templateand Adapter Primer (provided by 3′RACE system) was used to synthesizethe first strand of cDNA. Then cDNA was amplified by using differentcombinations of degenerate primers. The reaction mixture comprised 2.5microL10×PCR buffer, 1.5 microL 25 mM MgCl₂, 1.5 microL 25 mM MgCl₂, 0.5microL 10 mM dNTP mix, 0.5 microL, 10 microM 3′ Primer, 0.5 microL AUAP(10 microM, provided by 3′ RACE system), 0.5 microL TaqDNA polymerase (5u/microL, Promega), 1 microL cDNA synthesis reaction and autoclaved,distilled water to 25 microL.

PCR was performed under the following conditions: The reaction wassubmitted to 94° C. for 3 minutes followed by 30 cycles of 94° C. for 30sec, 50° C. for 30 sec and extension at 72° C. for 1 minute. A finalextension step at 72° C. for 10 minutes followed by a 4° C. hold stepcompleted the program.

PCR products of the right size for each pair of primer were recoveredfrom 1% agarose (1×TBE) gel, then purified by incubation in a 60° C.water bath followed by purification using GFXTMPCR DNA and Gel BandPurification Kit. (Amersham Pharmacia Biotech Inc., Cat. No.27-9602-01). The concentrations of purified products were determined bymeasuring the absorbance of A260 and A280 in a spectrophotometer. Thenthese purified fragments were ligated to pGEM-T Vector (Promega, Cat.No. A3600) according to the kit from Promega (Cat. No. A3600).

Using the “heat shock” method I microL ligation products weretransformed into 50 microL JM109 high efficiency competent cells.Transformation cultures were plated onto LB plates withampicillin/IPTG/X-Gal, and plates were incubated overnight at 37° C.Recombinant clones were identified by color screening on indicatorplates and colony PCR screening. The positive clones were inoculatedinto 3 ml LB liquid medium and incubate overnight at 37° C. on a rotaryshaker at 250 rpm. The cells were pelleted by centrifugation for 5 minat 10,000×g and plasmid sample were prepared from the cell pellet byusing Minipreps DNA Purification System (Promega, Cat. No. A7100).Finally the plasmids were sequenced with BigDye Terminator CycleSequencing Ready Reaction Kit (PE) by using ABI377 sequencer. Thesequencing reaction was as follows: 4 microL Terminator Ready ReactionMix, 1.0-1.5 microgram Plasmid DNA, 3.2 pmol Primer and dH₂O to a finalvolume of 10 microL.

Sequence analysis of the cDNA clones from different primer pairs showedthat the sequences contain coding regions of CBHII gene. The primerswere successfully used for molecular screening of CBHII gene from alltested fungal species within Chaetomium thermophilum, Myceliophtorathermophila, Acremonium thermophilum, Melanocarpus sp., Thielaviamicrospore, Aspergillus sp., Thielavia australiensis, Aspergillustubingensis, Gloeophyllum trabeum, Meripilus giganteus, Trichophaeasaccata, Stibella anualata and Malbrancheae cinnamonea. The identifiedCBH II encoding DNA sequences are shown as SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13,SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO:23 and SEQ ID NO: 25. Full-length sequences were obtained fromAspergillus tubingensis, Chaetomium thermophilum, Myceliophtorathermophila, Trichophaea saccata, Stibella anualata, and Malbrancheaecinnamonea. From Acremonium thermophilum, Melanocarpus sp., Thielaviamicrospore, Aspergillus sp., Thielavia australiensis, Gloeophyllumtrabeum and Meripilus giganteus only partial sequences have beenobtained.

Alternatively to the method applied above, the cDNA library could bescreened for the full-length cDNA using standard hybridizationtechniques and the partial cDNA sequence as a probe. The clones giving apositive hybridization signal with the probe are then purified andsequenced to determine the longest cDNA sequence. Homology search andcomparison confirms that the full-length cDNA correspond to the partialCBH II cDNA sequence that was originally used as a probe.

The two approaches described above rely on the presence of thefull-length CBH II cDNA in the cDNA library or in the cDNAs used for itsconstruction. Alternatively, the 5′ and 3′ RACE (Rapid Amplification ofcDNA Ends) techniques or derived techniques could be used to identifythe missing 5′ and 3′ regions. For this purpose, mRNAs from are isolatedand utilized to synthesize first strand cDNAs using oligo(dT)-containingAdapter Primer or a 5′-Gene Specific Primer (GSP).

The full-length cDNA of the CBH II gene can also be obtained by usinggenomic DNA. The CBH II gene can be identified by PCR techniques such asthe one describe above or by standard genomic library screening usinghybridization techniques and the partial CBH II cDNA as a probe.Homology search and comparison with the partial CBH II cDNA is used tothat the genomic sequence correspond to the CBH II gene. Identificationof consensus sequences such as initiation site of transcription, startand stop codons or polyA sites could be used to define the regioncomprising the full-length cDNA. Primers constructed from both the 5′and 3′ ends of this region could then be used to amplify the full-lengthcDNA from mRNA or cDNA library (see above).

By expression of the full-length gene in a suitable expression hostconstruct the CBH II enzyme is harvested as an intra cellular or extracellular enzyme from the culture broth.

Example 2

Using Blast the protein sequences were compared to SWall, ERDBP, andGenSeqP. If the sequenced was full length, the catalytic core waspredicted using PFAMM HMM and only that core region was used to searchthe databases. The highest hit to the public databases are listed exceptwhere the sequence is a duplicate to a sequence already present in theERDB.

Chaetomium thermophilum NP000980 has 83% identity to the Humicolainsolens avicelase II Glycosyl hydrolase in SWALL:Q9C1S9, family 6domain.

Myceliophtora thermophila NP001130 has 79% protein identity to the H.insolens NCE2 in geneseqp|aaw44827|aaw44827.

Acremonium sp. T178-4 NP001132 has 74% protein identity to theAcremonium cellulolyticus cellulase geneseqp|aaw25789|aaw25789. Glycosylhydrolase family 6 domain.

Melanocarpus sp. AT181-3 NP001133 has 91% protein identity to the H.insolens Cel6A fungal cellulase in geneseqp|aaw01077|aaw01077. Glycosylhydrolase family 6 domain.

Thielavia microspora T046-1 NP001134 has 79% protein identity to the H.insolens cellulase NC2 in geneseqp|aaw44827|aaw44827. Glycosyl hydrolasefamily 6 domain.

Aspergillus sp. T1882 NP001136 has 71% protein identity to theexocellobiohydrolase in geneseqp|aaw02321|aaw02321 Phanerochaetechrysosporium. Glycosyl hydrolase family 6 domain.

Thielavia australiensis NP001000 has 77% protein identity to the H.insolens cellulase NC2 protein in geneseqp|aaw44827|aaw44827. Glycosylhydrolase family 6 domain.

Aspergillus tubingensis NP001143 has 67% protein identity to the CBHIIin SWALL:Q8NIB5 Talaromyces emersonii. The DNA sequence entry is 94%identical to NP001144 Gloeophyllum trabeum. Glycosyl hydrolase family 6domain.

Gloeophyllum trabeum NP001144 67% protein identity to the CBHIISWALL:Q8NIB5 Talaromyces emersonii. The DNA sequence entry is 94%identical to NP001144 Gloeophyllum trabeum.

Example 3 Sequencing of the Malbranchia cinnamonea CBH II Gene and theStilbella anulata CBH II Gene

The cDNA inserts in plasmids Clone ZY043193, a cDNA encoding theMalbranchia cinnamonea CBH II, and clone ZY040206, a cDNA encoding theStilbella anulata CBH II, were sequenced to phred quality values >40,indicating high confidence DNA sequence data. DNA sequencing wasperformed on an ABI 3700 (ABI, Foster City, Calif.) according to themanufacturer's protocols. Assembly of sequence data was performed usingphred/phrap/consed (University of Washington).

Example 4 Construction of Expression Plasmids for the Malbranchiacinnamonea CBH II Gene and the Stilbella anulata CBH II Gene

The clone and the nucleotide sequences of the Malbranchia cinnamonea CBHII gene described above are used for subcloning of the gene andexpression in Aspergillus host. Polymerase chain reaction approach isused to subclone the CBHII gene (without its own promoter) from theisolated cDNA clone ZY043193 using primers designed from the nucleotidesequences. In order to facilitate the subcloning of the gene fragmentinto the pAILo 2 expression vector, BspHI and Pac I restriction enzymesites, respectively, at the 5′ and 3′ end of the gene, are introduced.The vector pAILo 2 contains the TAKA promoter, NA2-tpi leader and AMGterminator as regulatory sequences. The plasmid also containsAspergillus nidulans pyrG gene as a selectable marker for fungaltransformations. The following primers are used for PCR amplificationprocess:

(SEQ ID NO: 33) Primer F4 (forward): 5′ GGGTCATGAGAGACTCTTTGTTCAC 3′(SEQ ID NO: 34) Primer R4 (reverse):5′ GGGTTAATTAATTAGAATGGGGGGTTGGCATTTC 3′

PCR is performed using Pwo polymerase (Boehringer Mannheim) according tothe manufacturers specifications. The PCR amplified product is gelisolated and cut with BspH I and Pac I enzymes and gel purified. Thepurified fragment is ligated to a pAILo 2 vector (already cut with Nco Iand Pac I) to get the plasmid pEJG100 in which the transcription of theM. cinnamonea CBH II gene is under the control of the TAKA promoter. Theplasmid, pEJG100, is transformed into E. coli Solopac Gold cells(Stratagene, La Jolla, Calif.) cells. E. coli transformants containingthe pEJG100 plasmid are isolated and plasmid DNA is prepared fortransformation and expression in Aspergillus.

The clone and the nucleotide sequences of the Stilbella anulata CBH IIgene described above are used for subcloning of the gene and expressionin an Aspergillus host. Polymerase chain reaction approach is used tosubclone the CBHII gene (without its own promoter) from the isolatedcDNA clone ZY040206 using primers designed from the nucleotidesequences. In order to facilitate the subcloning of the gene fragmentinto the pALLO2 expression vector, PCR primers were designed containingrestriction sites compatible to the cloning sites of pALLO2 (NcoI andPacI) and to satisfy overlap requirements for the Infusion PCR kitprotocol (Clonetech, Palo Alto, Calif.). The following primers are usedfor PCR amplification process:

(SEQ ID NO: 35) Primer F4.1 (forward):5′ ACTGGATTTACCATGGCCGGTCGATTCTTCC 3′ (SEQ ID NO: 36) Primer R4.1(reverse): 5′ AGTCACCTCTAGTTATTAGAAGGCGGGGTTG 3′

The PCR product was generated using Pfx enzyme (Life Technologies) with1× enhancer. The 1400 bp product was gel excised, purified with Qiaquick(Qiagen, Valencia, Calif.), ligated into pALLO2 with the Infusionreaction. The resulting plasmid, pEJG96, is transformed into E. coliSolopac Gold cells (Stratagene, La Jolla, Calif.). E. coli transformantscontaining the pEJG96 plasmid are isolated and plasmid DNA is preparedfor transformation and expression in Aspergillus.

Example 5 Transformation of Aspergillus oryzae

Protoplasts are prepared from A. oryzae strain JAL 250 in which the pyrGgene of the host strain is deleted. Protoplast preparation andtransformation are done as previously described (Christensen et al.,supra). A. oryzae transformants expressing orotidine monophosphatedecarboxylase are selected based on their ability to grow in the absenceof uracil, Transformants are, spore purified twice on selective platesand the spore purified transformants used for further analysis.

Example 6

Expression of Malbranchia cinnamonea CBH II Gene and the Stilbellaanulata CBH II in A. oryzae

The transformants are screened for CBH II expression in shake flasks (25ml medium in 125 ml flasks) using a medium that contains the followingin g/L: maltose 50; MgSO₄.7H₂O, 2.0; KH₂PO₄, 10.0; K₂SO₄, 2.0; citricacid, 2.0; yeast extract, 10.0; AMG trace metal solution, 0.5 ml; urea2.0. The pH of the medium is adjusted to 6.5 before sterilization byautoclaving. Flasks are inoculated with freshly harvested spores andincubated in a shaker (200 rpm) at 34° C. Culture supernatants areharvested at 5 days. Five microliters of the culture supernatant is runon 8-16% Tris-Glycine gels. For the Malbranchia cinnamonea CBH II, thepredicted molecular weight of the protein is 43 kDa. A smear,significant over background, runs at about 50 kDa is seen in thetransformants. For the Stilbella anulata CBH II, the predicted molecularweight of the protein is 49 kDa. A band, significant over background,runs at about 55 kDa in the transformant.

Example 7

The phosphoric acid cellulose (PASC) was prepared as described bySchulein, 1997, J. Biotechnol. 57: 71-81. Protein concentrations weredetermined using a BCA Protein Assay (Pierce) as per the manufacturer'sinstructions. Protein aliquots were examined on 8-16% Acrylamidegradient gels (Invitrogen) and stained with Biosafe Coomassie Stain(Biorad).

Aspergillus oryzae broths expressing the Stilbella annulata Cel6A (˜55kD) and the Malbranchia cinnamonea Cel6A(˜49 kD) enzymes wereconcentrated using Centricon Plus 20 (Millipore) filtering devices usinga swinging bucket rotor centrifuge (Sorvall RC3B Plus; total time of ˜25minutes at 3000 rpm). Approximately 3 ml of each concentrate was loadedonto a 10DG Econo PAC column (Biorad) equilibrated with 50 mM sodiumacetate pH 5.0 and the desalted material eluted with 4 ml of 50 mMsodium acetate, pH 5.0. The protein concentrations for each sample weredetermined and aliquots analyzed on 8-16% Acrylamide gradient gels. APASC activity assay (endpoint assay) was performed utilizing a 96 wellmicroplate format. Briefly, 10 microL of appropriately diluted glucosestandards (2 mg/ml to 0.25 mg/ml) were placed in wells containing 190microL of 50 mM sodium acetate buffer pH 5.0 and 0.5 mg/ml BSA (Dilutionbuffer). Reagent controls (200 microL Dilution buffer), Sample controls(10 microL dilution to be assayed plus 190 microL Dilution buffer) andSubstrate controls (10 microL Dilution buffer plus 190 microL 2 g/L PASCin Dilution buffer) were included in each assay. A set of serialdilutions were generated for each sample to be assayed and 10 microL ofeach dilution placed in their designated wells. Reactions were initiatedby the addition of 190 microL of 2 g/L PASC. Samples were mixed and theplates placed in a 50° C. water bath for 30 minutes. The reactions werestopped by the addition of 500 microL of 0.5 M NaOH to each well. Plateswere centrifuged (Sorvall RT7) for 5 minutes at 2000 rpm and 100 microLaliquots of each sample transferred to a 96 well microtiter plate withconical wells. Determination of reducing sugar content was initiated byadding 50 microL of 1.5% (w/v) p-Hydroxybenzoic Acid Hydrazide (PHBAH)to each well and incubating the plate at 95° C. for 10 minutes. Theplate was allowed to cool to room temperature and 50 microL of doubledistilled H₂O added to each well. At this time 100 microL aliquots fromeach well were transferred to a flat bottomed 96 well microtiter plateand the OD 410 read using a Spectra MAX plate reader.

The glucose standards prepared for the PHBAH portion of the assay wereused to construct a glucose standard curve (A410 vs Glucoseconcentration in mg/ml). The slope and intercept from this standardcurve was used to generate a second graph in which the micromolesreducing sugar/min/ml was plotted vs protein concentration (mg/ml) togive the specific activities (IU/mg) of the samples assayed at 50° C.The specific activity for Stilbella annulata was 0.24 (IU/mg) and forMalbranchia cinnamonea 1.40 (IU/mg).

Example 8 Cellobiohydrolase Activity

A cellobiohydrolase is characterized by the ability to hydrolyze highlycrystalline cellulose very efficiently compared to other cellulases.Cellobiohydrolase may have a higher catalytic activity using PASC(phosphoric acid swollen cellulose) as substrate than using CMC assubstrate. For the purposes of the present invention, any of thefollowing assays can be used to identify cellobiohydrolase activity:

Activity on Azo-Avicel

Azo-Avicel (Megazyme, Bray Business Park, Bray, Wicklow, Ireland) wasused according to the manufacturer's instructions.

Activity on PNP-Beta-Cellobiose

50 microL CBH substrate solution (5 mM PNP beta-D-Cellobiose(p-Nitrophenyl β-d-Cellobioside Sigma N-5759) in 0.1 M Na-acetatebuffer, pH 5.0) was mixed with 1 mL substrate solution and incubated 20minutes at 40° C. The reaction was stopped by addition of 5 mL stopreagent (0.1 M Na-carbonate, pH 11.5). Absorbance was measured at 404nm.

Activity on PASC and CMC

The substrate is degraded with cellobiohydrolase to form reducingsugars. A Microdochium nivale carbohydrate oxidase (rMnO) or anotherequivalent oxidase acts on the reducing sugars to form H₂O₂ in thepresence of O₂. The formed H₂O₂ activates in the presence of excessperoxidase the oxidative condensation of 4-aminoantipyrine (AA) andN-ethyl-N-sulfopropyl-m-toluidine (TOPS) to form a purple product whichcan be quantified by its absorbance at 550 nm.

When all components except cellobiohydrolase are in surplus, the rate ofincrease in absorbance is proportional to the cellobiohydrolaseactivity. The reaction is a one-kinetic-step reaction and may be carriedout automatically in a Cobas Fara centrifugal analyzer (Hoffmann LaRoche) or another equivalent spectrophotometer which can measure steadystate kinetics.

Buffer: 50 mM Na-acetate buffer (pH 5.0);

Reagents: rMnO oxidase, purified Microdochium nivale carbohydrateoxidase, 2 mg/L

-   -   Peroxidase, SIGMA P-8125 (96 U/mg), 25 mg/L    -   4-aminoantipyrine, SIGMA A-4382, 200 mg/L    -   TOPS, SIGMA E-8506, 600 mg/L    -   PASC or CMC (see below), 5 g/L

All reagents were added to the buffer in the concentrations indicatedabove and this reagent solution was mixed thoroughly.

50 microL cellobiohydrolase II sample (in a suitable dilution) was mixedwith 300 microL reagent solution and incubated 20 minutes at 40° C.Purple color formation was detected and measured as absorbance at 550nm.

The AA/TOPS-condensate absorption coefficient is 0.01935 A₅₅₀/(microMcm). The rate is calculated as micromoles reducing sugar produced perminute from OD₅₅₀/minute and the absorption coefficient.

PASC:

Materials: 5 g Avicel® (Art. 2331 Merck);

-   -   150 mL 85% Ortho-phosphoric-acid (Art. 573 Merck);    -   800 mL Acetone (Art. 14 Merck);    -   Approx. 2 liters deionized water (Milli-Q);    -   1 L glass beaker;    -   1 L glass filter funnel;    -   2 L suction flask;    -   Ultra Turrax Homogenizer.

Acetone and ortho-phosphoric-acid is cooled on ice. Avicel® is moistedwith water, and then the 150 mL icecold 85% Orthophosphoric-acid isadded. The mixture is placed on an icebath with weak stirring for onehour.

Add 500 mL ice-cold acetone with stirring, and transfer the mixture to aglass filter funnel and wash with 3×100 mL ice-cold acetone, suck as dryas possible in each wash. Wash with 2×500 mL water (or until there is noodor of acetone), suck as dry as possible in each wash.

Re-suspend the solids in water to a total volume of 500 mL, and blend tohomogeneity using an Ultra Turrax Homogenizer. Store wet in refrigeratorand equilibrate with buffer by centrifugation and re-suspension beforeuse.

CMC:

Bacterial cellulose microfibrils in an impure form were obtained fromthe Japanese foodstuff “nata de coco” (Fujico Company, Japan). Thecellulose in 350 g of this product was purified by suspension of theproduct in about 4 L of tap water. This water was replaced by freshwater twice a day for 4 days.

Then 1% (w/v) NaOH was used instead of water and the product wasre-suspended in the alkali solution twice a day for 4 days.Neutralization was done by rinsing the purified cellulose with distilledwater until the pH at the surface of the product was neutral (pH 7).

The cellulose was microfibrillated and a suspension of individualbacterial cellulose microfibrils was obtained by homogenization of thepurified cellulose microfibrils in a Waring blender for 30 min. Thecellulose microfibrils were further purified by dialyzing thissuspension through a pore membrane against distilled water and theisolated and purified cellulose microfibrils were stored in a watersuspension at 4° C.

Example 9 Expression of Malbranchia Cinnamonea CBH II Gene in A. oryzae

The Malbranchia cinnamonea CBH II gene was expressed in Aspergillusoryzae and an enzyme of approximately 42 kDa was purified to a purity of95%. The activity was 1650 pnp-BDG.

Example 10

Two recombinantly expressed (Aspergillus oryzae) CBHII enzymes fromStilbella annulata (Cel6A) and Malbranchea cinnamonea (Cel6B) wereassayed for enzymatic activity on phosphoric acid cellulose (PASC).

Aspergillus oryzae broths expressing recombinant Stilbella annulataCel6A (˜55 kDa) and the Malbranchia cinnamonea Cel6B (˜49 kDa) wereconcentrated using Centricon Plus 20 filtering devices using a swingingbucket rotor (Sorvall RC3B Plus; ˜25 minutes at 3,000 rpm).Approximately 3 ml of each concentrate was loaded onto a 10DG Econo PACcolumn (Biorad) equilibrated with 50 mM sodium acetate pH 5.0 and thedesalted material eluted with 4 ml of 50 mM sodium acetate pH 5.0. Theprotein concentrations for each sample were determined using a BCAProtein Assay Kit (Pierce) and aliquots analyzed on 8-16% Acrylamidegradient gels (Invitrogen).

A PASC activity assay was performed utilizing a 96 well microplateformat. Briefly, 10 microL of an appropriate glucose standard (2 mg/mlto 0.25 mg/ml) was placed in a well containing 190 microL of 50 mMsodium acetate buffer pH 5.0 and 0.5 mg/ml BSA (Dilution buffer).Reagent controls (200 microL dilution buffer), sample controls (10microL dilution to be assayed plus 190 microL dilution buffer) andsubstrate controls (10 microL dilution buffer plus 190 microL 2 g/L PASCin dilution buffer) were also run. A series of serial dilutions were setup for each sample and 10 microL of each dilution placed in theirdesignated wells. Reactions were initiated by adding 190 microL of 2 g/LPASC. Plates were covered and placed in a 50° C. water bath for 30minutes. Reactions were stopped by the addition of 500 microL of 0.5 MNaOH to each well. Plates were centrifuged (Sorvall RT7) for 5 minutesat 2000 rpm. Approximately 100 microL aliquots of each sample weretransferred to a 96 well microtiter plate with conical wells. Each wellthen received 50 microL of 1.5% p-Hydroxybenzoic Acid Hydrazide (PHBAH)and was mixed thoroughly. Plates were incubated at 95° C. for 10minutes. Following the incubation step plates were cooled to roomtemperature and 50 microL of ddH₂O added to each well. One hundredmicroL aliquots from each well were transferred to flat bottomed 96 wellmicrotiter plates and the OD 410 nm read using a Spectra MAX platereader.

Using the glucose standard curve (A410 vs Glucose in mg/ml) generatedfor the PASC assay the slope and intercept from this curve was used toconstruct a second graph in which the umoles reducing sugar/min/ml wasplotted vs protein concentration (mg/ml) to give the specific activities(IU/mg) for the enzyme samples assayed. In determining specific activity(SA) on PASC only percent conversions of less than 2% were used.

Hydrolysis of PCS was conducted using 1.1 ml Immunoware microtubes(Pierce) using a total reaction volume of 1.0 ml. In this protocolhydrolysis of PCS (20 mg/ml in 50 mM sodium acetate pH 5.0 buffer) wasperformed using different protein loadings (expressed as mg Enzyme pergram PCS) of a Thielavia terrestris broth or Celluclast 1.5 L sample inthe presence of 3% Aspergillus oryzae beta glucosidase (3% of Cellulaseprotein loading). Characterization of Thielavia's PCS hydrolyzingcapability was done at multiple temperatures: 40° C., 50° C. and 65° C.(Isotemp 102S water baths). Typically, reactions were run in duplicateand aliquots taken during the course of hydrolysis (t=0, 2, 4, 6, 8 and24 hours). PCS hydrolysis reactions were stopped by mixing a 20 microLaliquot of each hydrolyzate with 180 microL of 0.44% NaOH (Stopreagent). Appropriate serial dilutions were generated for each sampleand the reducing sugar content determined using a p-Hydroxybenzoic AcidHydrazide (PHBAH) assay adapted to a 96 well microplate format. Briefly,a 90 microL aliquot of an appropriately diluted sample was placed in a96 well conical bottomed microplate. Reactions were initiated by adding60 microL of 1.5% (w/v) PHBAH in 2% NaOH to each well. Plates wereheated uncovered at 95° C. for 10 minutes. Plates were allowed to coolto RT and 50 microL of ddH₂O added to each well. A 100 microL aliquotfrom each well was transferred to a flat bottomed 96 well plate and theabsorbance at A410 nm measured using a SpectraMax Microplate Reader(Molecular Devices). Glucose standards (0.1-0.0125 mg/ml diluted with0.4% sodium hydroxide) were used to prepare a standard curve totranslate the obtained A410 values into glucose equivalents. Theresultant equivalents were used to calculate the percentage of PCScellulose conversion for each reaction. Our benchmark conditions forCelluclast 1.5 L PCS hydrolysis was the following: 50 mg/ml PCS in 50 mMsodium acetate pH 5.0, ˜21 mg Enzyme/g PCS (Equal to ˜10 FPU), in theabsence of externally added beta glucosidase at 38° C.

Aspergillus oryzae broths expressing the CBHII enzymes from Stilbellaannulata (Cel6A) and Malbranchea cinnamonea (Cel6B) were desalted,concentrated and their protein concentrations determined as described inthe materials and methods. Analysis of these recombinant protein sampleson a 8-16% Acrylamide gradient gel indicates the Stilbella Cel6A enzyme(FIG. 1, lane #1) has an apparent molecular weight of −55° kDa whilethat of Malbranchea Cel6B is ˜49 kDa (FIG. 1, lane #2).

To determine whether or not these recombinant enzymes were enzymaticallyactive hydrolysis reactions were conducted using a PASC substrate. Underthe conditions described previously Stilbella annulata (Cel6A) andMalbranchea cinnamonea (Cel6B) had specific activities of 0.24 IU/mg and1.40 IU/mg, respectively.

DEPOSIT OF BIOLOGICAL MATERIAL

China General Microbiological Culture Collection Center (CGMCC)

The following biological material has been deposited Dec. 19, 2002 underthe terms of the Budapest Treaty with the China General MicrobiologicalCulture Collection Center (CGMCC), Institute of Microbiology, ChineseAcademy of Sciences, Haidian, Beijing 100080, China:

Accession Number: CGMCC 0859

Applicants' reference: NP000980

Description: Chaetomium thermophilum

Classification: Chaetomiaceae, Sordariales, Ascomycota

Related sequence(s): SEQ ID NO: 1, SEQ ID NO: 2

Accession Number: CGMCC 0862

Applicants' reference: NP 001130

Description: Myceliophthora thermophila

Classification: Chaetomiaceae, Sordariales, Ascomycota

Related sequence(s): SEQ ID NO: 3, SEQ ID NO: 4

Accession Number: Acremonium sp. T178-4 CGMCC 0857

Applicants' reference: NP001132

Description: Acremonium sp. T178-4

Classification: mitosporic Ascomycetes

Related sequence(s): SEQ ID NO: 5, SEQ ID NO: 6

Accession Number: Melanocarpus sp. CGMCC 0861

Applicants' reference: NP001133

Description: Melanocarpus sp.

Classification: Trichocomaceae, Eurotiales, Ascomycota

Related sequence(s): SEQ ID NO: 7, SEQ ID NO: 8

Accession Number Thielavia microspora CGMCC 0863

Applicants' reference: NP001134

Description: Thielavia microspora

Classification: Chaetomiaceae, Soradariales, Ascomycota

Related sequence(s): SEQ ID NO: 9, SEQ ID NO: 10

Accession Number: Aspergillus sp. T186-2 CGMCC 0858

Applicants' reference: NP001132

Description: Aspergillus sp. T186-2

Classification: Trichocomaceae, Eurotiales, Ascomycota

Related sequence(s): SEQ ID NO: 11, SEQ ID NO: 12

Accession Number: Thielavia austfaliensis CGMCC 0864

Applicants' reference: NP001000

Description: Thielavia australiensis

Classification: Chaetomiaceae, Sordariales, Ascomycota

Related sequence(s): SEQ ID NO: 13, SEQ ID NO: 14

American Type Culture Collection (ATCC)

The following biological material is obtainable from American TypeCulture Collection, P.O. Box 1549, Manassas, Va. 20108, USA.

Accession Number: ATCC 11.39

Applicants reference: NP001144

Description: Gloeophyllum trabeum

Classification: -

Related sequence(s): SEQ ID NO: 17, SEQ ID NO: 18

Centraalbureau Voor Schimmelcultures (CBS)

The following biological material is obtainable from Centraalbureau VoorSchimmelcultures (CBS), Uppsalalaan 8, 3584 CT Utrecht, The Netherlands(alternatively P.O. Box 85167, 3508 AD Utrecht, The Netherlands):

Accession Number: CBS 161.79

Applicants' reference: NP001143

Description: Aspergillus tubingensis

Classification: -

Related sequence(s): SEQ ID NO: 15, SEQ ID NO: 16

Accession Number: CBS 521.95

Applicants' reference: ND001631

Description: Meripilus giganteus

Classification: -

Related sequence(s): SEQ ID NO: 19, SEQ ID NO: 20

Accession Number: CBS 804.70

Applicants' reference: NP000960

Description: Trichophaea saccata

Classification: -

Related sequence(s): SEQ ID NO: 21, SEQ ID NO: 22

Accession Number: CBS 185.70

Applicants' reference: NP001040

Description: Stilbella annulata

Classification: -

Related sequence(s): SEQ ID NO: 23, SEQ ID NO: 24

Accession Number: CBS 115.68

Applicants' reference: NP 001045

Description: Malbranchea cinnamomea

Classification: -

Related sequence(s): SEQ ID NO: 25, SEQ ID NO: 26

1. An isolated polypeptide having cellobiohydrolase II activity,selected from the group consisting of: (a) a polypeptide having an aminoacid sequence comprising the amino acid sequence shown as amino acids 1to 82 of SEQ ID NO:4, and a polypeptide having an amino acid sequencecomprising at least 85% identity with the amino acid sequence shown asamino acids 1 to 420 of SEQ ID NO: 4; (b) a polypeptide having an aminoacid sequence which is encoded by nucleotides 1 to 246 of SEQ ID NO: 3and nucleotides 1 to 1272 of SEQ ID NO: 3; (c) a polypeptide which isencoded by a nucleotide sequence which hybridizes under high stringencyconditions with a polynucleotide probe selected from the groupconsisting of: (i) the full complementary strand of the nucleotidesselected from the group consisting of: nucleotides 1 to 246 of SEQ IDNO: 3, and nucleotides 1 to 1272 of SEQ ID NO: 3; and (ii) the fullcomplementary strand of the nucleotides selected from the groupconsisting of: nucleotides 1 to 200 of SEQ ID NO: 3, and nucleotides 1to 1272 of SEQ ID NO: 3; and (d) a fragment of (a), (b) or (c) that hascellobiohydrolase II activity, wherein said high stringencyhybridization of the nucleotide sequence is at 42° C. in 5×SSPE, 1.0%SDS, 5×Denhardt's solution, 100 micro gram/ml denatured salmon spermDNA, and followed by washing three times each for 15 minutes using0.1×SSC, 0.1% SDS at 60° C.
 2. The polypeptide of claim 1, comprising anamino acid sequence selected from the group consisting of: a polypeptidehaving an amino acid sequence selected from the group consisting of: anamino acid sequence comprising the amino acid sequence shown as aminoacids 1 to 82 of SEQ ID NO: 4, and a polypeptide having an amino acidsequence which has at least 90% identity with the amino acid sequenceshown as amino acids 1 to 420 of SEQ ID NO:
 4. 3. The polypeptide ofclaim 1, where the polypeptide is a variant which comprises an aminoacid sequence that has at least one substitution, deletion or insertionof an amino acid, which is 90% identical to amino acids 1 to 420 of SEQID NO:
 4. 4. A detergent composition comprising a surfactant and thepolypeptide of claim 1.